Journal: bioRxiv

Article Title: Short-term oxaliplatin exposure according to established hyperthermic intraperitoneal chemotherapy (HIPEC) protocols lacks effectiveness in vitro and ex vivo

doi: 10.1101/709055

Figure Lengend Snippet: Normalized cell index (nCI; normalized to 1 at treatment) from RTCA measurements at three days (72 hours) after short-term incubation with hyperthermia and OCS ( post interventionem ; p.i.) obtained from patients during HIPEC. From left to right: (+ ctrl.): Triton; (-ctrl.): Physioneal 40; pre (circulated solvent [Physioneal] without addition of OX), and OCS sampled 10, 20 and 30 minutes after addition of OX to the circuit during HIPEC treatment. Pat. 1-9 are depicted from left to right as already characterized in . At HIPEC initiation OCS samples contained a theoretical average OX dosage of 90 µg/ml (≙ 227 µM). Panel (a) shows effects after 30 minutes incubation at 42 °C with respective OCS. Panel (b) shows respective effects after 60 minutes OCS incubation at 42 °C. Colored arrows mark conditions, when an average decrease in impedance of >50 % of the initial values (LC 50 ) was reached 72 hours after treatment. Graphs show mean ± SD (of 2-5 replicates). The LC 50 threshold is marked with a black line. A decrease of nCI values signifies cell death of OAW-42 target cells. Respective normalized cell index (nCI) readings in 6-hour intervals of RTCA measurements are provided in as well as Suppl. Fig. 1-14 .

Article Snippet: HIPEC conditions were subsequently simulated by incubation of cells for 30 or 60 minutes at 42 °C in an ambient air incubation shaker (Infors, Bottmingen, Switzerland) with slight movement (50 rotations per minute).

Techniques: Incubation, Solvent

Journal: bioRxiv

Article Title: Short-term oxaliplatin exposure according to established hyperthermic intraperitoneal chemotherapy (HIPEC) protocols lacks effectiveness in vitro and ex vivo

doi: 10.1101/709055

Figure Lengend Snippet: Normalized cell index (nCI) in 6-hour intervals from RTCA measurements of platinum-sensitive OAW-42 cells, incubated with OCS (0 h) at 42 °C, previously obtained from patients during HIPEC for the indicated periods and drug solvent circulated through the abdomen sampled before drug application (pre). OCS was obtained at time points 5, 10, 15, 20, 25 and 30 minutes (min.) after drug addition to the HIPEC circuit. Controls represent: (+ ctrl.): Triton; (-ctrl.): Physioneal 40 or medium (MEM). Impedance values (nCI) of samples obtained during HIPEC from Pat. 3 incubated for 30 minutes (a) and 60 minutes (b) , as well as from Pat. 1 after incubation for 30 minutes (c) and 60 minutes (d) are shown. The duration until 50 % impedance decrease (LC 50 ) was reached with 30/ 60 minutes exposure or continuous incubation of respective samples is given (e) . The samples from patients (pre + OCS) correspond to the material previously characterized and values of continuous incubation were taken from Löffler et al. . This material contains a theoretical maximum average OX dosage of 90 µg/ml (≙ 227 µM), however for continuous exposure samples had to be diluted by 50 % with serum supplemented medium due to technical reasons. Values were normalized to 1 at the start of treatment (0 h). The red box highlights values three days (72 hours) after treatment (see also ). A decrease of nCI values signifies cell death of OAW-42 target cells. Graphs show mean ± SD (of 2-4 replicates). Respective normalized cell index (nCI) readings in 6-hour intervals of RTCA measurements after incubation with HIPEC sample materials obtained from Pat. 2 and 4-9 are provided in Suppl. Fig. 1-14 .

Article Snippet: HIPEC conditions were subsequently simulated by incubation of cells for 30 or 60 minutes at 42 °C in an ambient air incubation shaker (Infors, Bottmingen, Switzerland) with slight movement (50 rotations per minute).

Techniques: Incubation, Solvent

Journal: bioRxiv

Article Title: Short-term oxaliplatin exposure according to established hyperthermic intraperitoneal chemotherapy (HIPEC) protocols lacks effectiveness in vitro and ex vivo

doi: 10.1101/709055

Figure Lengend Snippet: Normalized cell index (nCI) in 6-hour intervals obtained from RTCA impedance measurements of platinum-sensitive OAW-42 cells incubated for 30 minutes at 42 °C with the specified amounts of OX spiked into dextrose 5 % in water (D5W) (a) or Physioneal 40 (PDS) (b) performed at time point 0 hours (h). (+ ctrl.): Triton; (-ctrl.): Physioneal 40; OX (oxaliplatin); PDS (peritoneal dialysis solution; Physioneal 40); RTCA (real-time cell analysis). Graphs show mean ± SD (of 2-3 replicates). The duration until 50 % impedance decrease (LC 50 ) was reached after 30 minutes exposure of OX spiked into PDS/ D5W or with continuous incubation of OX spiked into D5W or PDS (together with 50 % serum supplemented medium) at the given concentrations is depicted (e) . Values were normalized to 1 at the start of treatment (0 h). The colored boxes highlight values three days (72 hours) after treatment. Respective nCI in 6-hour intervals obtained from RTCA impedance measurements of platinum-sensitive OAW-42 cells incubated continuously with OX are provided as Suppl. Fig. 17 & 18 .

Article Snippet: HIPEC conditions were subsequently simulated by incubation of cells for 30 or 60 minutes at 42 °C in an ambient air incubation shaker (Infors, Bottmingen, Switzerland) with slight movement (50 rotations per minute).

Techniques: Incubation, Cell Analysis

Journal: bioRxiv

Article Title: Short-term oxaliplatin exposure according to established hyperthermic intraperitoneal chemotherapy (HIPEC) protocols lacks effectiveness in vitro and ex vivo

doi: 10.1101/709055

Figure Lengend Snippet: Platinum-sensitive OAW-42 cells and the colon carcinoma cell line HT29 were seeded at a density of 3.15 × 10 5 cells per well (in a 24-well plate) and incubated for 30 minutes at 42 °C with the specified amounts of OX spiked either into dextrose 5 % in water (D5W) (a) , or into PDS (b) . Further, cells were incubated continuously with the specified amounts of OX spiked into culture medium (MEM) to allow for continuous drug exposure and heated likewise initially (i.e. 30 minutes incubation at 42 °C followed by 72 hours at 37 °C) (c) . After OX exposure, cells were washed and subsequently cultured in serum supplemented medium for another three days or remained with drugs in culture medium (MEM). Afterwards, a fluorometric resazurin-based (CellTiter-Blue ® , CTB) ( left graph ) and a sulforhodamine B (SRB) assay ( right graph ) was used to determine cell viability. Cells were normalized to cells treated identically with D5W/ PDS/ MEM only (viability ctrl.). (+ ctrl.): Triton. Graphs show mean ± SD from three independent experiments each performed in triplicates. The LC 50 threshold is marked with a black line.

Article Snippet: HIPEC conditions were subsequently simulated by incubation of cells for 30 or 60 minutes at 42 °C in an ambient air incubation shaker (Infors, Bottmingen, Switzerland) with slight movement (50 rotations per minute).

Techniques: Incubation, Cell Culture, Sulforhodamine B Assay

Journal: bioRxiv

Article Title: Short-term oxaliplatin exposure according to established hyperthermic intraperitoneal chemotherapy (HIPEC) protocols lacks effectiveness in vitro and ex vivo

doi: 10.1101/709055

Figure Lengend Snippet: Platinum-sensitive OAW-42 cells were incubated with 5 % dextrose in water (D5W) or with peritoneal dialysis solution (PDS) at 42 °C for 30 minutes (a) or 60 minutes (b) and compared to untreated cells cultured in medium (MEM). Histograms from flow cytometry showing cell counts vs. forward scatter area (FSC-A) ( left panel ) and a comparison of FSC-A between the different conditions ( right panel ). Significant differences are marked by an asterisk (Bonferroni corrected Student’s t -test).

Article Snippet: HIPEC conditions were subsequently simulated by incubation of cells for 30 or 60 minutes at 42 °C in an ambient air incubation shaker (Infors, Bottmingen, Switzerland) with slight movement (50 rotations per minute).

Techniques: Incubation, Cell Culture, Flow Cytometry, Comparison